Biosensor

ABSTRACT

The present invention provides a biosensor having a favorable response characteristic up to a high concentration range, a low blank response and a high storage stability. This sensor comprises an electrode system including a working electrode and a counter electrode, for forming an electrochemical measurement system by coming in contact with a supplied solution; an electrically insulating supporting member for supporting the electrode system; a first reagent layer formed on the working electrode; and a second reagent layer formed on the counter electrode, wherein the first reagent layer comprises an enzyme as the main component, and the second reagent layer comprises an electron mediator as the main component.

BACKGROUND OF THE INVENTION

The present invention relates to a biosensor for rapid quantification ofa substrate contained in a sample with high accuracy.

Conventionally, methods using polarimetry, colorimetry, reductimetry anda variety of chromatography have been developed as the measure forquantitative analysis of sugars such as sucrose and glucose. However,those conventional methods are all poorly specific to sugars and hencehave poor accuracy. Among them, the polarimetry is simple inmanipulation, but it is largely affected by the temperature during themanipulation. Therefore, this method is not suitable for simplequantification of sugars at home by ordinary people.

In recent years, a variety of biosensors have been developed which bestutilize a specific catalytic action of enzymes.

In the following, a method of quantitative analysis of glucose will beexplained as an example of the method for quantifying a substratecontained in a sample. Conventionally known electrochemicalquantification of glucose includes a method using a combination ofglucose oxidase (EC 1.1.3.4: hereinafter abbreviated to “GOD”) as anenzyme with an oxygen electrode or a hydrogen peroxide electrode (see“Biosensor” ed. by Shuichi Suzuki, Kodansha, for example).

GOD selectively oxidizes β-D-glucose as a substrate toD-glucono-δ-lactone using oxygen as an electron mediator. Oxygen isreduced to hydrogen peroxide during the oxidation reaction by GOD in thepresence of oxygen. A decreased volume of oxygen is measured by theoxygen electrode, or an increased volume of hydrogen peroxide ismeasured by the hydrogen peroxide electrode. The decreased volume ofoxygen or, otherwise, the increased volume of hydrogen peroxide isproportional to the content of glucose in the sample. It is thereforepossible to quantify glucose based on the decreased volume of oxygen orthe increased volume of hydrogen peroxide.

In the above method, it is possible to quantify glucose in the sampleaccurately by using the specificity of the enzyme reaction. However, asspeculated from the reaction, this prior art method has a drawback thatthe measurement result is greatly affected by the oxygen concentrationin the sample. Hence, in the event where oxygen is absent in the sample,measurement is infeasible.

Under such a circumstance, a glucose sensor of new type has beendeveloped which uses as the electron mediator an organic compound or ametal complex such as potassium ferricyanide, a ferrocene derivative anda quinone derivative, in place of oxygen in the sample. The sensor ofthis type oxidizes the reduced electron mediator resulting from theenzyme reaction on a working electrode so as to determine the glucoseconcentration in the sample based on an oxidation current produced bythe oxidation reaction. At this time, on a counter electrode, theoxidized electron mediator is reduced, and a reaction for generating thereduced electron mediator proceeds. With the use of such an organiccompound or metal complex as the electron mediator in place of oxygen,it is possible to form a reagent layer by precisely placing a knownamount of GOD together with the electron mediator in their stable stateon the electrode, thereby enabling accurate quantification of glucosewithout being affected by the oxygen concentration in the sample. Inthis case, it is also possible to integrate the reagent layer containingthe enzyme and electron mediator with an electrode system while keepingthe reagent layer in an almost dry state, and therefore a disposableglucose sensor based on this technology has recently been notedconsiderably. A typical example of such a glucose sensor is a biosensordisclosed in Japanese Laid-Open Patent Publication Hei 3-202764. Withsuch a disposable glucose sensor, it is possible to measure the glucoseconcentration easily with a measurement device by simply introducing asample into the sensor connected detachably to the measurement device.The application of such a technique is not limited to quantification ofglucose and may be extended to quantification of any other substratecontained in the sample.

However, in the above-described conventional biosensors, when the samplecontains a substrate at high concentrations, the enzyme reactionproceeds also on the counter electrode and supply of the electronmediator to the counter electrode thus becomes insufficient, so that thereaction at the counter electrode becomes a rate determining step, whichmakes it impossible to obtain a current response proportional to thesubstrate concentration. Therefore, such biosensors have a problem thatquantification of a substrate is not possible when the sample contains asubstrate at high concentrations.

In recent years, there is a demand for a biosensor exhibiting a lowresponse when the substrate concentration is zero and excellent storagestability. The response obtained when the substrate concentration iszero is hereinafter referred to as “blank response”.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a biosensor comprising: an electrodesystem including a working electrode and a counter electrode, forforming an electrochemical measurement system by coming in contact witha supplied sample solution; an electrically insulating supporting memberfor supporting the electrode system; a first reagent layer formed on theworking electrode; and a second reagent layer formed on the counterelectrode, wherein the first reagent layer comprises an enzyme as themain component, and the second reagent layer comprises an electronmediator as the main component.

It is preferred that the first reagent layer does not contain anelectron mediator and that the second reagent layer does not contain anenzyme.

In a preferred mode of the present invention, the supporting membercomprises an electrically insulating base plate on which the workingelectrode and the counter electrode are formed.

In another preferred mode of the present invention, the supportingmember comprises an electrically insulating base plate and anelectrically insulating cover member for forming a sample solutionsupply pathway or a sample solution storage section between the covermember and the base plate, the working electrode is formed on the baseplate, and the counter electrode is formed on an inner surface of thecover member so as to face the working electrode.

It is preferred that the cover member comprises a sheet member having anoutwardly expanded curved section, for forming a sample solution supplypathway or a sample solution storage section between the cover memberand the base plate.

In a more preferred mode of the present invention, the cover membercomprises a spacer having a slit for forming the sample solution supplypathway and a cover for covering the spacer.

It is preferred that at least the first reagent layer contains ahydrophilic polymer.

While the novel features of the invention are set forth particularly inthe appended claims, the invention, both as to organization and content,will be better understood and appreciated, along with other objects andfeatures thereof, from the following detailed description taken inconjunction with the drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a vertical cross-sectional view of a glucose sensor accordingto one example of the present invention.

FIG. 2 is an exploded perspective view of the glucose sensor, omittingthe reagent layers and surface active agent layer therefrom.

FIG. 3 is a vertical cross-sectional view of a glucose sensor accordingto another example of the present invention.

FIG. 4 is an exploded perspective view of the glucose sensor, omittingthe reagent layers and surface active agent layer therefrom.

FIG. 5 is a vertical cross-sectional view of a glucose sensor accordingto still another example of the present invention.

FIG. 6 is an exploded perspective view of the glucose sensor, omittingthe reagent layers and surface active agent layer therefrom.

FIG. 7 is a vertical cross-sectional view of a glucose sensor of acomparative example.

DETAILED DESCRIPTION OF THE INVENTION

A biosensor in accordance with a preferred mode of the present inventioncomprises an electrically insulating base plate; a working electrode anda counter electrode formed on the base plate; a first reagent layerformed on the working electrode; and a second reagent layer formed onthe counter electrode, wherein the first reagent layer comprises anenzyme as the main component, and the second reagent layer comprises anelectron mediator as the main component.

In this biosensor, the enzyme reaction hardly proceeds at the counterelectrode especially when the sample contains a substrate at highconcentrations, since the main component of the second reagent layer onthe counter electrode is the electron mediator. Thus, the probability ofcollision between the enzyme and the electron mediator decreases in thesample solution in which the reagents are dissolved, so that thelinearity of response current is decreased. However, since sufficientelectron mediator is retained on the counter electrode for the reaction,the reaction at the counter electrode does not become a rate determiningstep. As a result, the linearity of response current can be maintainedeven up to a high substrate-concentration range.

A biosensor in accordance with another preferred mode of the presentinvention comprises an electrically insulating base plate; anelectrically insulating cover member for forming a sample solutionsupply pathway or a sample solution storage section between the covermember and the base plate; a working electrode formed on the base plate;a counter electrode formed on an inner surface of the cover member so asto face the working electrode; a first reagent layer formed on theworking electrode; and a second reagent layer formed on the counterelectrode.

The cover member comprises a sheet member having an outwardly expandedcurved section, for forming a sample solution supply pathway or a samplesolution storage section between the cover member and the base plate.

A more preferred cover member comprises a spacer with a slit for formingthe sample solution supply pathway and a cover for covering the spacer.

In such a biosensor, since the first reagent layer and second reagentlayer are formed on separate members, respectively, the first reagentlayer and second reagent layer having different compositions can bereadily separated from each other. Moreover, since the working electrodeand counter electrode are formed at opposite positions, the ion tranferbetween the electrodes is facilitated, thereby further increasing thecurrent response.

In a biosensor whose cover member comprises the spacer and cover, sincethe physical strength of the cover is enhanced, the first reagent layerand second reagent layer are not brought into contact with each other byan external physical pressure, thereby preventing degradation in theenzyme activity due to the contact between the enzyme and the electronmediator.

In either of the biosensors of the above-described embodiments, it ispreferred that at least the first reagent layer contains a hydrophilicpolymer. Since the hydrophilic polymer prevents adsorption of proteins,etc. to the working electrode, the current response sensitivity isfurther improved. Besides, during the measurement, since the viscosityof a sample solution is increased by the hydrophilic polymer dissolvedin the sample solution, the effects of physical impact, etc. on thecurrent response are reduced, thereby improving the stability of thecurrent response.

In the present invention, for the base plate, spacer and cover, it ispossible to use any material having an insulating property andsufficient rigidity during storage and measurement. Examples of such amaterial include thermoplastic resins such as polyethylene, polystyrene,polyvinyl chloride, polyamide and saturated polyester resin, orthermosetting resins such as a urea resin, melamine resin, phenol resin,epoxy resin and unsaturated polyester resin. Among these resins,polyethylene terephthalate is preferred in view of the adhesiveness tothe electrode.

For the working electrode, it is possible to use any conductive materialif it is not oxidized itself in oxidizing the electron mediator. For thecounter electrode, it is possible to use a generally used conductivematerial such as palladium, silver, platinum, and carbon.

As the enzyme, it is possible to use the one suitable for the type of asubstrate in the sample, which is the subject of measurement. Examplesof the enzyme include fructose dehydrogenase, glucose oxidase, alcoholoxidase, lactate oxidase, cholesterol oxidase, xanthine oxidase, andamino acid oxidase.

Examples of the electron mediator include potassium ferricyanide,p-benzoquinone, phenazine methosulfate, methylene blue, and ferrocenederivatives. Besides, even when oxygen is used as the electron mediator,a current response is obtained. These electron mediators are used singlyor in combinations of two or more.

A variety of hydrophilic polymers are applicable. Examples of thehydrophilic polymer include hydroxyethyl cellulose, hydroxypropylcellulose, methyl cellulose, ethyl cellulose, ethylhydroxyethylcellulose, carboxymethyl cellulose, polyvinyl pyrrolidone, polyvinylalcohol, polyamino acid such as polylysine, polystyrene sulfonate,gelatin and its derivatives, polyacrylic acid and its salts,plolymethacrylic acid and its salts, starch and its derivatives, and apolymer of maleic anhydride or a maleate. Among them, carboxymethylcellulose, hydroxyethyl cellulose and hydroxypropyl cellulose areparticularly preferred.

The following description will explain the present invention in furtherdetail by illustrating examples thereof.

EXAMPLE 1

A glucose sensor will be explained as an example of a biosensor.

FIG. 1 is a vertical cross-sectional view of a glucose sensor of thisexample, and FIG. 2 is an exploded perspective view of the glucosesensor, omitting the reagent layers and surface active agent layertherefrom.

First, a silver paste was printed on an electrically insulating baseplate 1 made of polyethylene terephthalate by screen printing to formleads 2 and 3 and the base of later-described electrodes. Then, aconductive carbon paste containing a resin binder was printed on thebase plate 1 to form a working electrode 4. This working electrode 4 wasin contact with the lead 2. Further, an insulating paste was printed onthe base plate 1 to form an insulating layer 6. The insulating layer 6covered the peripheral portion of the working electrode 4 so that afixed area of the working electrode 4 was exposed. Next, a counterelectrode 5 was formed by printing a conductive carbon paste containinga resin binder so as to be in contact with the lead 3.

A first aqueous solution containing GOD as an enzyme and no electronmediator was dropped on the working electrode 4 of the base plate 1 andthen dried to form a first reagent layer 7. Besides, a second aqueoussolution containing potassium ferricyanide as an electron mediator andno enzyme was dropped on the counter electrode 5 of the base plate 1 andthen dried to form a second reagent layer 8. Further, in order toachieve smooth supply of a sample, a layer 9 containing lecithin as asurface active agent was formed so as to cover the first reagent layer 7and the second reagent layer 8.

Finally, the base plate 1, a cover 12 and a spacer 10 were adhered toeach other in a positional relationship as shown by the dashed lines inFIG. 2 to fabricate the glucose sensor.

The spacer 10 to be inserted between the base plate 1 and the cover 12has a slit 11 for forming a sample solution supply pathway between thebase plate 1 and the cover 12.

Since an air vent 14 of the cover 12 communicates with this samplesolution supply pathway, when the sample is brought into contact with asample supply port 13 formed at an open end of the slit 11, the samplereadily reaches the first reagent layer 7 and second reagent layer 8 inthe sample solution supply pathway because of capillary phenomenon.

As a comparative example, a glucose sensor was fabricated in the samemanner as this example with the exception of the process of forming thereagent layers. FIG. 7 is a vertical cross-sectional view of the glucosesensor of the comparative example. A reagent layer 30 was formed bydropping an aqueous solution containing GOD and potassium ferricyanideon the working electrode 4 and counter electrode 5 and then drying theaqueous solution. Moreover, a layer 9 containing lecithin as a surfaceactive agent was formed on the reagent layer 30.

Next, with the glucose sensors of Example 1 and the comparative example,the concentration of glucose was measured using a solution containing acertain amount of glucose as a sample. The sample was supplied to thesample solution supply pathway from the sample supply port 13 and, afterelapse of a certain time, a voltage of 500 mV was applied to the workingelectrode 4 using the counter electrode 5 as reference. Since the spacer10 is interposed between the cover 12 and the base plate 1, the strengthof the sensor against an external physical pressure is increased.Consequently, the volume of the sample solution supply pathway isreadily kept constant, and the effects of physical pressure, etc. on thecurrent response are reduced.

The value of a current which flowed across the working electrode 4 andthe counter electrode 5 upon the application of this voltage wasmeasured. As a result, in both of Example 1 and the comparative example,a current response proportional to the glucose concentration in thesample was observed. When the sample comes into contact with the firstreagent layer 7 and the second reagent layer 8, potassium ferricyanideas the oxidized form of the electron mediator dissociates intoferricyanide ion and potassium ion. The glucose in the sample, theferricyanide ion dissolved in the sample from the second reagent layer 8and the GOD react with each other. As a result, the glucose is oxidizedinto glucono lactone, and the oxidized form ferricyanide ion is reducedto the reduced form ferrocyanide ion. A reaction of oxidizingferrocyanide ion into ferricyanide ion proceeds on the working electrode4, while a reaction of reducing ferricyanide ion into ferrocyanide ionproceeds on the counter electrode 5. Since the concentration offerrocyanide ion is proportional to the concentration of glucose, it ispossible to measure the concentration of glucose based on the oxidationcurrent of the ferrocyanide ion.

In the glucose sensor of this example, high linearity was observed up toa higher glucose concentration than in the glucose sensor of thecomparative example for the following reasons.

Since GOD and potassium ferricyanide are separately carried on theworking electrode and the counter electrode, respectively, the enzymereaction hardly proceeds at the counter electrode. Thus, sufficientconcentration of potassium ferricyanide is retained on the counterelectrode, preventing the reaction at the counter electrode frombecoming a rate determining step even when the sample contains asubstrate at high concentrations. As a result, it is possible tomaintain the linearity of response current up to a high concentrationrange. In the sensor of the comparative example, on the other hand, theenzyme reaction proceeds on the counter electrode to the extent which isalmost equivalent to that on the working electrode, causing reduction offerricyanide ion into ferrocyanide ion. Consequently, the ferricyanideion becomes insufficient for the reaction on the counter electrode, sothat the reaction at the counter electrode becomes a rate determiningstep.

Further, in the glucose sensor of this example, the blank response waslowered and the current response was not changed so much even after along-time storage in comparison with the glucose sensor of thecomparative example. This is because GOD and potassium ferricyanide wereseparated from each other so that it was possible to prevent contact andinteraction between GOD and potassium ferricyanide, thereby suppressingan increase in the blank response and degradation in the enzyme activityduring a long-time storage.

EXAMPLE 2

FIG. 3 is a vertical cross-sectional view of a glucose sensor of thisexample, and FIG. 4 is an exploded perspective view of the glucosesensor, omitting the reagent layers and surface active agent layertherefrom.

A working electrode 4 and a lead 2 were formed by sputtering palladiumon an electrically insulating base plate 21. Next, by pasting aninsulating sheet 23 on the base plate 21, the working electrode 4 and aterminal section to be inserted into a measurement device were defined.

Meanwhile, a counter electrode 5 was formed by sputtering palladium ontothe inner wall surface of an outwardly expanded curved section 24 of anelectrically insulating cover member 22. An end portion of the curvedsection 24 was provided with an air vent 14.

A first aqueous solution containing GOD as an enzyme and no electronmediator was dropped on the working electrode 4 of the base plate 21 andthen dried to form a first reagent layer 7. Besides, a second aqueoussolution containing potassium ferricyanide as an electron mediator andno enzyme was dropped on the counter electrode 5 of the cover member 22to form a second reagent layer 8. Further, a layer 9 containing lecithinas a surface active agent was formed on the first reagent layer 7.

Finally, the base plate 21 and cover 22 were adhered to each other tofabricate the glucose sensor. Accordingly, the working electrode 4 andthe counter electrode 5 are positioned to face each other with a spaceformed between the base plate 21 and the curved section 24 of the covermember 22 therebetween. This space serves as a sample storage sectionand, when a sample is brought into contact with an open end of thespace, the sample readily moves toward the air vent 14 due to capillaryphenomenon and comes into contact with the first reagent layer 7 andsecond reagent layer 8.

Next, the concentration of glucose was measured according to the sameprocedure as in Example 1. As a result, a current response proportionalto the concentration of glucose in the sample was observed. The counterelectrode 5 was electrically connected by holding an end portion of thecurved section 24 with a clip connected to a lead wire.

In the glucose sensor of Example 2, the second reagent layer 8 containedonly potassium ferricyanide, so that high linearity was observed up to ahigher glucose concentration than in the glucose sensor of thecomparative example, like Example 1. Also, in this biosensor, likeExample 1, the blank response was lowered, and the current response wasnot changed so much even after a long-time storage in comparison withthe comparative example, since GOD and potassium ferricyanide wereseparated from each other. Further, in comparison with the comparativeexample, an increase in the response value was observed, because theworking electrode 4 and counter electrode 5 were formed at oppositepositions so that ion transfer between the electrodes was facilitated.

EXAMPLE 3

FIG. 5 is a vertical cross-sectional view of a glucose sensor of thisexample, and FIG. 6 is an exploded perspective view of the glucosesensor, omitting the reagent layers and surface active agent layertherefrom.

First, a silver paste was printed on an electrically insulating baseplate 31 made of polyethylene terephthalate by screen printing to form alead 2. Then, a conductive carbon paste containing a resin binder wasprinted on the base plate 31 to form a working electrode 4. This workingelectrode 4 was in contact with the lead 2. Further, an insulating pastewas printed on the base plate 31 to form an insulating layer 6. Theinsulating layer 6 covered the peripheral portion of the workingelectrode 4 so that a fixed area of the working electrode 4 was exposed.

Next, a silver paste was printed on the inner surface of an electricallyinsulating cover 32 to form a lead 3, and then a conductive carbon pastewas printed to form a counter electrode 5. Further, an insulating pastewas printed to form an insulating layer 6. The cover 32 was providedwith an air vent 14.

A first aqueous solution containing GOD as an enzyme and no electronmediator was dropped on the working electrode 4 of the base plate 31 andthen dried to form a first reagent layer 7, while a second aqueoussolution containing potassium ferricyanide as an electron mediator andno enzyme was dropped on the counter electrode 5 of the cover 32 andthen dried to form a second reagent layer 8. Further, a layer 9containing lecithin as a surface active agent was formed on the firstreagent layer 7.

Finally, the base plate 31, the cover 32 and a spacer 10 were adhered toeach other in a positional relationship as shown by the dashed lines ofFIG. 6 to fabricate the glucose sensor.

The spacer 10 interposed between the base plate 31 and the cover 32 hasa slit 11 for forming a sample solution supply pathway between the baseplate 31 and the cover 32. The working electrode 4 and counter electrode5 are positioned to face each other in the sample solution supplypathway formed in the slit 11 of the spacer 10.

Since the air vent 14 of the cover 32 communicates with this samplesolution supply pathway, when a sample is brought into contact with asample supply port 13 formed at an open end of the slit 11, the samplereadily reaches the first reagent layer 7 and second reagent layer 8 inthe sample solution supply pathway because of capillary phenomenon.

Next, the concentration of glucose was measured according to the sameprocedure as in Example 1. As a result of the measurement, a currentresponse proportional to the concentration of glucose in the sample wasobserved.

In the glucose sensor of Example 3, the second reagent layer 8 containedonly potassium ferricyanide, so that high linearity was observed up to ahigher glucose concentration than in the glucose sensor of thecomparative example, like Example 1. Also, since the working electrode 4and the counter electrode 5 were formed at opposite positions, thecurrent response was increased in comparison with the comparativeexample, like Example 2.

Moreover, since the GOD and potassium ferricyanide were separated fromeach other, like Example 1, the blank response was lowered and thecurrent response was not changed so much even after a long-time storagein comparison with the comparative example.

Furthermore, since the spacer 10 was interposed between the base plate31 and cover 32, the strength of the sensor against an external physicalpressure was enhanced. As a result, the first reagent layer 7 and thesecond reagent layer 8 were never brought into contact with each otherby the physical pressure, thereby preventing the current response frombeing varied by the degradation of the enzyme activity caused by thecontact between GOD and potassium ferricyanide. In addition, since thevolume of the sample solution supply pathway was readily kept constant,the stability of the current response was improved in comparison withExample 2.

EXAMPLE 4

In this embodiment, a glucose sensor was fabricated in the same manneras in Example 3 with the exception of the process of forming the firstreagent layer 7 and second reagent layer 8.

A first aqueous solution containing GOD as an enzyme, carboxymethylcellulose as a hydrophilic polymer and no electron mediator was droppedon the working electrode 4 of the base plate 31 and then dried to form afirst reagent layer 7, while a second aqueous solution containingpotassium ferricyanide as an electron mediator, carboxymethyl celluloseand no enzyme was dropped on the counter electrode 5 of the cover 32 andthen dried to form a second reagent layer 8. Moreover, the layer 9containing lecithin as a surface active agent was formed on the firstreagent layer 7.

Next, the concentration of glucose was measured according to the sameprocedure as in Example 1. As a result of the measurement, a currentresponse proportional to the concentration of glucose in the sample wasobserved.

In the glucose sensor of this example, the second reagent layer 8contained only potassium ferricyanide, so that high linearity wasobserved up to a higher glucose concentration than in the glucose sensorof the comparative example, like Example 1. Also, since the workingelectrode 4 and the counter electrode 5 were formed at oppositepositions, an increase in the response value was observed in comparisonwith the comparative example.

Moreover, since the GOD and potassium ferricyanide were separated fromeach other, the blank response was lowered and the current response wasnot changed so much even after a long-time storage in comparison withthe comparative example.

Furthermore, since the spacer 10 was interposed between the base plate31 and cover 32, it was possible to prevent the current response frombeing varied by the degradation in the enzyme activity caused by thecontact between GOD and potassium ferricyanide. In addition, since thevolume of the sample solution supply pathway was readily kept constant,the stability of the current response was improved in comparison withExample 2.

Besides, in comparison with Examples 2 and 3, the current response wasfurther increased for the following reason. The presence ofcarboxymethyl cellulose in the first reagent layer 7 preventedadsorption of proteins to the surface of the working electrode 4, andhence the electrode reaction on the working electrode 4 proceededsmoothly. Furthermore, since the viscosity of the sample was increasedduring the measurement, the effects of physical impact, etc. on thesensor were reduced and variations in the sensor response weredecreased.

In the above-described examples, while a voltage of 500 mV was appliedto the working electrode 4 using the counter electrode 5 as reference,the voltage is not necessarily limited to 500 mV. Any voltage thatenables oxidation of the electron mediator reduced with the enzymereaction may be applied.

In the foregoing examples, the second reagent layer formed on thecounter electrode contained only the electron mediator, but it maycontain other components than the electron mediator as long as theinclusion of such components does not make the reaction at the counterelectrode a rate determining step and the influence it may have on theblank response and storage stability is so small as to be negligible.Also, in those examples, the first reagent layer formed on the workingelectrode contained either only the enzyme or the enzyme and thehydrophilic polymer, but it may also contain the other components aslong as the influence such inclusion may have on the blank response andstorage stability is so small as to be negligible.

In the above-described examples, only one kind of electron mediator wasused, but two or more kinds of electron mediators may be used.

The first reagent layer 7 and second reagent layer 8 may be immobilizedon the working electrode 4 or the counter electrode 5 so as toinsolubilize the enzyme or the electron mediator. In the case where thefirst reagent layer 7 and second reagent layer 8 are immobilized, it ispreferable to use a crosslinking immobilization method or an adsorptionmethod. Alternatively, the electron mediator and the enzyme may be mixedinto the working electrode and the counter electrode, respectively.

As the surface active agent, it is possible to use a material other thanlecithin. Besides, in the above-described examples, although the surfaceactive agent layer 9 was formed only on the first reagent layer 7, or onthe first reagent layer 7 and second reagent layer 8, the formation ofthe surface active agent layer 9 is not necessarily limited to theseexamples, and the surface active agent layer 9 may be formed at aposition facing the sample solution supply pathway, such as a side faceof the slit 11 of the spacer 10.

In the above-described examples, a two-electrode system consisting onlyof the working electrode and counter electrode is described. However, ifa three-electrode system including an additional reference electrode isadopted, it is possible to perform a more accurate measurement.

It is preferred that the first reagent layer 7 and the second reagentlayer 8 are not in contact with each other and are separated from eachother with a space interposed therebetween. Accordingly, it is possibleto further enhance the effect of suppressing an increase in the blankresponse and the effect of improving the storage stability.

As described above, according to the present invention, it is possibleto obtain a biosensor having a favorable current response characteristicup to a high concentration range. Further, it is possible to obtain abiosensor having a low blank response and a high storage stability.

Although the present invention has been described in terms of thepresently preferred embodiments, it is to be understood that suchdisclosure is not to be interpreted as limiting. Various alterations andmodifications will no doubt become apparent to those skilled in the artto which the present invention pertains, after having read the abovedisclosure. Accordingly, it is intended that the appended claims beinterpreted as covering all alterations and modifications as fall withinthe true spirit and scope of the invention.

What is claimed is:
 1. A biosensor comprising: an electrode systemincluding a working electrode and a counter electrode, for forming anelectrochemical measurement system by coming in contact with a suppliedsample solution; an electrically insulating supporting member forsupporting said electrode system; a first reagent layer formed on saidworking electrode; and a second reagent layer formed on said counterelectrode, wherein said first reagent layer comprises an enzyme as themain component, and said second reagent layer comprises an electronmediator as the main component, and wherein said supporting membercomprises an electrically insulating base plate and an electricallyinsulating cover member for forming a sample solution supply pathway ora sample solution storage section between said cover member and saidbase plate, said working electrode is formed on said base plate, andsaid counter electrode is formed on an inner surface of said covermember so as to face said working electrode.
 2. The biosensor inaccordance with claim 1, wherein the first reagent layer does notcontain an electron mediator, and the second reagent layer does notcontain an enzyme, and said working electrode oxidizes a reduced form ofsaid electron mediator.
 3. The biosensor in accordance with claim 1,wherein said cover member comprises a sheet member having an outwardlyexpanded curved section, for forming a sample solution supply pathway ora sample solution storage section between said cover member and saidbase plate.
 4. The biosensor in accordance with claim 1, wherein saidcover member comprises a spacer having a slit for forming said samplesolution supply pathway and a cover for covering said spacer.
 5. Thebiosensor in accordance with claim 1, wherein said first reagent layercontains a hydrophilic polymer.